Uridine-cytidine kinase has been purified 1800-fold from the mouse mast cell ascites tumor P815, to a specific activity of 3.02 international units/mg enzyme protein. The final preparation is also highly stable. This preparation has been characterized with respect to divalent cation requirement, pH optimum, and specificity of both phosphate donor and phosphate acceptor substrates. Specificity for the nucleoside triphosphate donor is broad, while that for the nucleoside substrate is quite narrow. Only uridine, cytidine, and their analogs are phosphorylated. Thymine riboside, deoxyribosides, and cytosine arabinoside are inactive as substrates. Sensitivity of the enzyme to feedback inhibition by the end-products CTP and UTP is retained throughout the purification procedure. The initial velocity pattern has been determined with the purified enzyme; the pattern rules out a ping-pong reaction mechanism for the enzyme and indicated that the reaction proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. Analysis of the data by Cleland's Sequen computer program has indicated a Km of the enzyme for uridine of 1.5 X 10 to the minus 4th power M, for cytidine of 4.5 X 10 to the minus 5 M, and for ATP, with either uridine or cytidine as phosphate acceptor, of 3.6 X 10 to the minus 3 M or 2.1 X 10 to the minus 3M, respectively. The V max was 1.83 microm moles phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 microm moles for the cytidine kinase reaction. Thus the rate of uridine phosphorylation was twice that for cytidine but the Km for cytidine was only one-third that for uridine.